Many methods for peptide synthesis are described in the literature (for example, see U.S. Pat. No. 6,015,881; Mergler et al. (1988) Tetrahedron Letters 29:4005-4008; Mergler et al. (1988) Tetrahedron Letters 29:4009-4012; Kamber et al. (eds), Peptides, Chemistry and Biology, ESCOM, Leiden (1992) 525-526; Riniker et al. (1993) Tetrahedron Letters 49:9307-9320; Lloyd-Williams et al. (1993) Tetrahedron Letters 49:11065-11133; and Andersson et al. (2000) Biopolymers 55:227-250. The various methods of synthesis are distinguished by the physical state of the phase in which the synthesis takes place, namely liquid phase or solid phase.
In solid phase peptide synthesis (SPPS), an amino acid or peptide group is bound to a solid support resin. Then, successive amino acids or peptide groups are attached to the support-bound peptide until the peptide material of interest is formed. The support-bound peptide is then typically cleaved from the support and subject to further processing and/or purification. In some cases, solid phase synthesis yields a mature peptide product; in other cases the peptide cleaved from the support (i.e., a “peptide intermediate fragment”) is used in the preparation of a larger, mature peptide product.
Peptide intermediate fragments generated from solid phase processes can be coupled together in the solid phase or in a liquid phase synthetic process (herein referred to as “solution phase synthesis”). Solution phase synthesis can be particularly useful in cases where the synthesis of a useful mature peptide by solid phase is either impossible or not practical. For example, in solid phase synthesis, longer peptides eventually may adopt an irregular conformation while still attached to the solid support, making it difficult to add additional amino acids or peptide material to the growing chain. As the peptide chain becomes longer on the support resin, the efficiency of process steps such as coupling and deprotection may be compromised. This, in turn, can result in longer processing times to compensate for these problems, in addition to incremental losses in starting materials, such as activatable amino acids, co-reagents, and solvents. These problems can increase as the length of the peptide increases.
Therefore, it is relatively uncommon to find mature peptides of greater than 30 amino acids in length synthesized in a single fragment using only a solid phase procedure. Instead, individual fragments may be separately synthesized on the solid phase, and then coupled in the solid and/or solution phase to build the desired peptide product. This approach requires careful selection of fragment candidates. While some general principles can guide fragment selection, quite often empirical testing of fragment candidates is required. Fragment strategies that work in one context may not work in others. Even when reasonable fragment candidates are uncovered, process innovations may still be needed for a synthesis strategy to work under commercially reasonable conditions. Therefore, peptide synthesis using hybrid schemes are often challenging, and in many cases it is difficult to predict what problems are inherent in a synthesis scheme until the actual synthesis is performed.
In solution phase coupling, two peptide intermediate fragments, or a peptide intermediate fragment (or “fragment”) and a reactive amino acid, are coupled in an appropriate solvent, usually in the presence of additional reagents that promote the efficiency and quality of the coupling reaction. The peptide intermediate fragments (or “fragment”) are reactively arranged so the N-terminal of one fragment becomes coupled to the C-terminal of the other fragment, or vice versa. In addition, side chain protecting groups, which are present during solid phase synthesis, are commonly retained on the fragments during solution phase coupling to ensure the specific reactivity of the terminal ends of the fragments. These side chain-protecting groups are typically not removed until a mature peptide has been formed.
Modest improvements in one or more steps in the overall synthetic scheme can amount to significant improvements in the preparation of the mature peptide. Such improvements can lead to a large overall saving in time and reagents, and can also significantly improve the purity and yield of the final product. Proper selection of chemical strategies is necessary for this hybrid approach as there are significant pitfalls due to poor solubility of fully protected fragments and due to the ease of epimerization in solution phase couplings.
While the discussion of the importance of improvements in hybrid synthesis is applicable to any sort of peptide produced using these procedures, it is of particular importance in the context of peptides that are therapeutically useful and that are manufactured on a scale for commercial medical use. Synthesis of larger biomolecular pharmaceuticals, such as therapeutic peptides, can be very expensive. Because of the cost of reagents, synthesis time, many synthesis steps, in addition to other factors, very small improvements in the synthetic process of these larger biomolecular pharmaceuticals can have a significant impact on whether it is even economically feasible to produce such a pharmaceutical. Such improvements are necessary due to these high production costs for larger biomolecular pharmaceuticals as supported by the fact that, in many cases, there are few, if any, suitable therapeutic alternatives for these types of larger biomolecular pharmaceuticals.
This is clearly seen in the case of insulinotropic peptides such as the Exenatide peptide and its functional counterparts. Such peptides are possible therapeutic agents for the treatment of type 2 non-insulin-dependent diabetes mellitus and obesity. The peptides improve the initial rapid release of endogenous insulin, suppress glucagon release of the pancreas, regulate gastric emptying, and reduce appetite—all of which function to lower blood glucose. Exenatide is self-regulating in that it lowers blood sugar when levels are elevated but does not continue to lower blood sugar when levels return to normal.
Native Exenatide is isolated from the Gila monster and is 39 amino acid residues long. Exenatide has a molecular weight of 4186.6 Daltons. Native Exenatide, referred to as exendin-4 when it is created synthetically, is commercially available under the trade designation BYETTA™ and may be represented by the notation Exenatide(1-39). This notation indicates that the peptide has all amino acids from 1 (N-terminus) through 39 (C-terminus). Exenatide has the amino acid sequence according to SEQ ID NO. 1:
His1-Gly2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9- Leu10-Ser11-Lys12-Gln13-Met14-Glu15-Glu16-Glu17- Ala18-Val19-Arg20-Leu21-Phe22-Ile23-Glu24-Trp25- Leu26-Lys27-Asn28-Gly29-Gly30-Pro31-Ser32-Ser33- Gly34-Ala35-Pro36-Pro37-Pro38-Ser39
A key challenge in the solid and solution phase synthesis of Exenatide relates to the sequence of three glutamic acid residues in the 15, 16 and 17 positions. Indeed, any peptide having at least two glutamic acid residues in sequence like this will tend to share this challenge. Specifically, it is difficult to chemically synthesize peptide fragments very far beyond such glutamic acid residues. Without wishing to be bound by theory, the repeating Glu sequence tends to yield a fragment portion that twists in the solid phase. This makes it relatively difficult to continue to build fragment size through the Glu chain effectively. In conventional practice, a fragment having a sequence of two or more repeating Glu residues might only be able to have 1 to 3 amino acids upstream (toward the C terminus) and/or downstream (toward the N-terminus) as a practical matter. The issue tends to be more severe downstream from the repeating Glu chain. This often may mean that peptide fragments grown in the solid phase that contain a sequence of repeating Glu residues tend to be relatively short.
In the case of Exenatide, this has impacted solid phase fragment strategy. In one instance, a fragment scission point might be positioned within the Glu sequence so that one fragment includes only one Glu residue. This strategy in the context of the Exenatide peptide, though, still leaves the other fragment with two Glu residues in a row and may dictate a four fragment synthesis strategy in the solid phase before fragments are coupled in the solution phase. Alternatively, all three Glu residues can be included in one fragment. However, using conventional strategies, this may mean that after the third Glu residue is added to the fragment under construction, it might only be practical to add the Met, the Met and Gln, or possibly the Met, Gln and Lys residues to that fragment, leaving the remaining residues to be assembled in a separate peptide fragment. Again, this may dictate a four fragment synthesis strategy in the solid phase before fragments are coupled in the solution phase. While a four fragment synthesis approach may be desirable in some instances, the conventional handling of the repeating Glu sequences makes even these strategies more problematic than would be desired.
In short, practical concerns associated with repeating Glu sequences may cause a synthesis strategy to resort to extra fragments to ensure that the fragments incorporating the repeating Glu residues are relatively short. It would be highly desirable to be able to use solid phase synthesis to synthesize longer fragments that include two or more Glu residues in a row.
In addition to these concerns, issues relating to product recovery and product purity for the large-scale production of peptides, as well as reagent handling, storage and disposal, can greatly impact the feasibility of the peptide synthesis scheme. Thus, there is a continuing need for peptide synthesis processes capable of efficiently producing peptide materials of commercial interest in large batch quantities with improved yields.